Multiple integrative vectors and Yarrowia lipolytica transformants

ABSTRACT

This invention relates to modified Yarrowia lipolytica LEU2 gene promoters; modified Y. lipolytica LEU2 genes comprising such modified Y. lipolytica LEU2 gene promoters; and vectors comprising such modified Y. lipolytica LEU2 genes. This invention also relates to vectors comprising a Y. lipolytica DNA sequence LEU2 genes. This invention also relates to vectors comprising a Y. lipolytica DNA sequence sufficient for integrative transformation of Y. lipolytica; vectors which comprise a nucleotide sequence coding for a polypeptide and a promoter functional in Y. lipolytica operably linked thereto; E. coli transformants which comprise vectors according to this invention; Y. lipolytica transormants which comprise an expression vector according to this invention; methods of producing Y. lipolytica transformants comprising multiple integrated expression vectors; strains of Y. lipolytica useful in the preparation of such transformants; methods of producing polypeptides with certain of the Y. lipolytica transformants; nucleotide sequences useful in the preparation of modified Y. lipolytica LEU2 gene promoters according to this invention and a method for producing modified Y. lipolytica LEU2 promoters.

This is a 371 of PCT/IB94/00128 which is a continuation of U.S. patentapplication Ser. No. 08/117,375 filed Sep. 2, 1993, now abandoned.

TECHNICAL FIELD

This invention relates to modified Yarrowia lipolytica LEU2 genepromoters and to modified Y. lipolytica LEU2 genes comprising suchmodified Y. lipolytica LEU2 gene promoters. This invention furtherrelates to vectors comprising such modified Y. lipolytica LEU2 genes.Such vectors include vectors comprising a Y. lipolytica DNA sequencesufficient for integrative transformation of Y. lipolytica at a locusother that the LEU2 locus of Y. lipolytica; and vectors which comprise anucleotide sequence coding for a polypeptide and a promoter functionalin Y. lipolytica operably linked thereto. The latter vectors are alsoknown as expression vectors. Further still, this invention relates to E.coli transformants comprising vectors of this invention.

This invention also relates to Y. lipolytica transformants whichcomprise an expression vector according to this invention; to methods ofproducing Y. lipolytica transformants comprising multiple integratedexpression vectors; to strains of Y. lipolytica useful in thepreparation of such transformants; and to methods of producingpolypeptides with certain of the Y. lipolytica transformants. Furtherstill, this invention relates to nucleotide sequences useful in thepreparation of modified Y. lipolytica LEU2 gene promoters according tothis invention and a method for producing modified Y. lipolytica LEU2promoters.

BACKGROUND ART

Processes for transformation of Y. lipolytica as well as vectors usefultherefor and transformants comprising such vectors, inter alia, aredisclosed and claimed in U.S. Pat. Nos. 4,880,741 and 5,071,764, both ofwhich are assigned to the assignee hereof. Vectors useful intransformation of Y. lipolytica for expression and secretion ofheterologous proteins as well as transformants and processes forproducing heterologous protein therewith, inter alia, are disclosed andclaimed in U.S. Pat. No. 4,937,189 which also is assigned to theassignee hereof. U.S. Pat. No. 4,937,189 also discloses the nucleotidesequence of the LEU2 gene of Y. lipolytica (SEQUENCE I.D. NO: 1).

WO91/00920 (PCT/EP90/01138), published Jan. 24, 1991, discloses aprocess for preparing a homologous or heterologous protein by a yeasttransformed by multicopy integration of an expression vector into thegenome of the yeast. The expression vector used in that process containsboth an "expressible gene" encoding the desired protein and a "deficientselection marker needed for the growth of the yeast in a specificmedium" such as a defective LEU2 gene (leu2d). Also disclosed are suchvectors which also contain DNA coding for ribosomal RNA.

WO92/01800 (PCT/US91/04899), published Feb. 6, 1992, disclosesintegrating plasmid vectors capable of inserting throughout the yeastgenome with high copy number and a process to accomplish suchintegration. The vectors disclosed use "dispersed repetitive elements(DRE's)" such as the yeast DELTA sequences, Ty sequences and tRNA DNAsequences.

DISCLOSURE OF THE INVENTION

This invention provides modified Y. lipolytica LEU2 gene promotersuseful in the preparation of modified Y. lipolytica LEU2 genes. Morespecifically, this invention provides modified Y. lipolytica LEU2 genepromoters selected from the group consisting of nucleotides 693 to 798of SEQUENCE I.D. NO: 1, nucleotides 718 to 798 of SEQUENCE I.D. NO: 1,nucleotides 718 to 798 of SEQUENCE I.D. NO: 1 wherein nucleotide 724 ischanged from A to G, nucleotides 718 to 798 of SEQUENCE I.D. NO: 1wherein nucleotide 725 is changed from T to G, nucleotides 718 to 798 ofSEQUENCE I.D. NO: 1 wherein nucleotide 722 is changed from A to G,nucleotides 745 to 798 of SEQUENCE I.D. NO: 1, and the functionalequivalents thereof. This invention also provides modified Y. lipolyticaLEU2 genes comprising a modified LEU2 gene promoter according to thisinvention functionally linked to a Y. lipolytica LEU2 structural genecoding sequence.

This invention further provides vectors comprising modified Y.lipolytica LEU2 genes as described hereinabove. Still further, thisinvention provides vectors comprising a modified Y. lipolytica LEU2 geneas described hereinabove and a Y. lipolytica DNA sequence sufficient forintegrative transformation of Y. lipolytica at a locus other than theLEU2 locus of Y. lipolytica. A preferred Y. lipolytica DNA sequence forsuch vectors is a Y. lipolytica ribosomal DNA sequence. Also provided bythis invention is a vector which is useful in the construction ofexpression vectors as described hereinbelow.

The vectors according to this invention comprising a Y. lipolytica DNAsequence sufficient for integrative transformation at a locus other thanthe LEU2 locus are capable, upon transformation, of integrating atmultiple sites and, hence, result in Y. lipolytica transformantscomprising multiple copies of such vectors.

The invention further provides expression vectors which, upontransformation of Y. lipolytica, result in both expression of aheterologous polypeptide and secretion thereof by said Y. lipolytica.Such expression vectors comprise a modified Y. lipolytica LEU2 gene (asdescribed hereinabove), a DNA sequence sufficient for integrativetransformation of Y. lipolytica at a locus other than the LEU2 locus ofY. lipolytica, an XPR2 promoter of Y. lipolytica and, operably linked tosuch a promoter, the signal, pro-1 or pro-2 sequence of the XPR2 gene ofY. lipolytica, or a functional fragment or equivalent thereof, which, inturn, is operably linked to a coding sequence for a polypeptide.Preferred are such expression vectors comprising the signal, pro-1 orpro-2 sequence of the XPR2 gene of Y. lipolytica wherein the DNAsequence sufficient for integrative transformation of Y. lipolytica at alocus other than the LEU2 locus is a Y. lipolytica ribosomal DNAsequence. Also preferred are such expression vectors or preferredexpression vectors wherein the polypeptide is a heterologouspolypeptide. Preferred heterologous polypeptides of this inventioninclude prochymosin, proinsulin analog, insulinotropin and human TGF-β3.

Still further, this invention provides Y. lipolytica transformantscomprising the expression vectors of this invention. Also provided bythis invention is a Y. lipolytica strain comprising a deletion in theLEU2 locus which strain is useful in the selection of modified LEU2genes according to this invention and E. coli transformants comprisingvectors of this invention.

Yet further still, this invention provides processes for producing apolypeptide which comprise fermenting a Y. lipolytica transformant ofthis invention in an aqueous nutrient medium comprising assimilablesources of carbon, nitrogen and inorganic salts.

Also provided by this invention is a method of producing Y. lipolyticatransformants comprising multiple integrated expression vectors. Themethod comprises transforming a Y. lipolytica strain having a deletionof the LEU2 gene thereof with an expression vector capable ofintegrative transformation as described hereinabove and which vector hasbeen linearized by cleaving the expression vector in the DNA sequencesufficient for integrative transformation, and selecting the bestgrowing transformants on a medium which lacks leucine.

A method of producing a modified Y. lipolytica LEU2 promoter is alsoprovided by this invention. The method comprises producing a DNAsequence having a 5' end and a 3' end, having homology or substantialhomology to a region of SEQUENCE I.D. NO: 1 and wherein said 5' end iswithin, but not co-terminus with the 5' end of the promoter region ofSEQUENCE I.D. NO: 1; producing a vector comprising (i) a DNA sequencewherein the 3' end of the DNA sequence produced as described immediatelyabove is joined to the 5' end of a DNA sequence comprising nucleotidesfrom SEQUENCE I.D. NO: 1 such that the structural gene for LEU2 isformed and (ii) a sequence coding for a second Y. lipolytica structuralgene; transforming a Y. lipolytica host having a deletion of the LEU2gene and a mutation or deletion in the structural gene corresponding tosaid second structural gene with the vector produced as describedimmediately above, which vector has been cleaved within the regioncoding for said second structural gene; selecting Y. lipolyticatransformants on a medium containing leucine but requiring said secondstructural gene for growth; and screening such transformants for atransformant which grows poorly on a medium lacking leucine. A preferredsecond structural gene for use in the above method is the URA3 gene ofY. lipolytica.

As used throughout this Specification and the appendant claims,"functional fragment" means a fragment of the sequence to which thephrase refers which fragment has a function which is at least part ofthe overall function of the complete sequence.

As used throughout this Specification and the appendant claims,"functional equivalent" means a sequence having the same, orsubstantially the same function as the sequence to which the phraserefers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are schematic representations of the construction of plasmidpNB276.

FIGS. 2A-2C are schematic representations of the construction of plasmidpNB243.

FIGS. 3A-3C are schematic representations of the construction of plasmidpNB308.

FIGS. 4A-4F are schematic representations of the construction of plasmidpMIVINS.

FIGS. 5A-5B are schematic representations of the construction of plasmidpNB747.

MATERIALS AND METHODS

E. coli strain DH5α (obtained as competent cells from Bethesda ResearchLaboratories, Gaithersburg, Md.) was used as host for plasmidpreparation. E. coli cells were transformed according to the supplier'srecommended procedure and grown on LB medium plus ampicillin (100 μg/ml)at 37° C. LB medium contained (per liter): 10 g Bactotryptone, 5 g Bactoyeast extract and 10 g sodium chloride. Plasmid DNA was prepared usingthe alkaline lysis method (Birnboim, H. C. et al., NAR 7, 1513 (1979) orthe boiling method (Holmes, D. S. et al., Anal. Biochem. 114:193(1981)).

Restriction endonucleases, T4 polymerase, T4 DNA ligase, Klenow DNApolymerase, calf intestinal alkaline phosphatase, as well othernecessary enzymes as indicated hereinbelow were purchased from NewEngland Biolabs (Beverly, Mass.) and/or Bethesda Research Laboratories(Gaithersburg, Md.) and were used according to the supplier'srecommended conditions. All molecular biological manipulations wereperformed according to standard methods (Sambrook, J., E. F. Fritsch andT. Maniatis, Molecular Cloning, A Laboratory Manual (2nd Ed.) CSHL Press(1989)).

Y. lipolytica strains were grown on standard rich yeast medium (YPD)containing 1% Bacto-yeast extract, 2% Bacto-peptone and 2% dextrose, oron complete minimal medium without leucine (Sherman, F., et al.,Laboratory Course Manual for Methods in Yeast Genetics, CSHL Press 1986)at 28° C. Transformation was performed as described by Davidow, L. S.,et al., Curr Genet 10: 39-48 (1985). For expression, cultures were grownon Medium A (5% Bacto-peptone, 1% glucose, 0.1% yeast extract). YeastDNA was prepared as described by Sherman, F., et al., Laboratory CourseManual for Methods in Yeast Genetics, CSHL Press (1986) and Southernanalysis was performed using standard conditions (Sambrook, J., E. F.Fritsch and T. Maniatis, Molecular Cloning, A Laboratory Manual (2ndEd.) CSHL Press 1989). The methods referenced above are well known tothose skilled in the art.

DETAILED DESCRIPTION

1. CONSTRUCTION OF MODIFIED Y. lipolytica LEU2 GENE PROMOTERS ANDMODIFIED Y. lipolytica LEU2 GENES.

The nucleotide sequence of the Y. lipolytica LEU2 gene is given in theSequence Listing, below, as SEQUENCE I.D. NO: 1. The Y. lipolytica LEU2gene nucleotide sequence is disclosed, inter alia, in U.S. Pat. No.4,937,189, which is assigned to the assignee hereof and which isincorporated herein by reference. Further, plasmid pLD25 containing theY. lipolytica LEU2 gene is disclosed in U.S. Pat. No. 5,071,764, anddisclosed and claimed in U.S. Pat. No. 4,880,741, both of which are alsoassigned to the assignee hereof and which are incorporated herein byreference. Plasmid pLD25 has been deposited with the American TypeCulture Collection, Rockville, Md., USA under the terms of the BudapestTreaty in the form of an E. coli transformant (E. coli JC-355transformant with pLD25) and has been designated ATCC 39464. Allrestrictions on availability of ATCC 39464 have been irrevocably removedby virtue of the grant of U.S. Pat. No. 4,880,741.

The construction of promoter-deleted LEU2 genes is presentedschematically in FIG. 1. Plasmid pNB258, which serves as a template forpolymerase chain reaction (PCR), includes a wild type Y. lipolytica URA3gene as a positive control for transformation efficiency and to providethe ability to test the promoter-deleted LEU2 genes for the ability tocomplement a leucine deficient host as a single copy. To constructpNB258, a 1.6 kb blunt-ended DNA fragment containing URA3 sequences wasisolated from pLD106 by digesting plasmid DNA with StuI and SalIrestriction enzymes, and treating the restriction digest with Klenowfragment according to supplier's instructions. Approximately 200 ng ofthis DNA was ligated with approximately 500 ng of SalI digested, Klenowblunted, alkaline phosphatase-treated pBluescript+SK vector (StratageneCloning Systems, La Jolla, Calif.) using T4 DNA ligase according tosupplier's directions. One-third of the ligation mix was transformedinto E. coli strain DH5-α, and plated on LB plates containing 100 μg/mlampicillin. DNA was prepared from selected single colony isolates oftransformants and digested with KpnI and BamHI restriction enzymes.Restriction fragments were separated on 1% agarose gels and visualizedby ethidium bromide staining. Plasmid pNB257 was identified based on thepresence therein of a 1.6 kb DNA fragment containing the URA3 gene.

A LEU2 gene was constructed in which an internal EcoRI site was removedby a single base change which did not alter the LEU2 coding sequences.This LEU2 gene construct allowed the isolation of the LEU2 gene as asingle EcoRI DNA fragment, which made further manipulations moreconvenient. This construct was generated as follows. DNA from plasmidpLD40 (which contains the LEU2 gene in pBR322 and is described in U.S.Pat. No. 4,880,741 and U.S. Pat. No. 5,071,764) was digested with XhoI,which removed a 29 base pair fragment of DNA containing an internalEcoRI site, and treated with alkaline phosphatase. Linear DNA wasseparated electrophoretically on a 0.7% agarose gel and electroeluted.Two hundred (200) ng of this linear DNA was ligated to 50 ng ofsynthetic linker DNA which contained a single base pair change (C/G toT/A at base 22) which removed the EcoRI site without changing thecorresponding LEU2 codon. The synthetic linker used was of the sequence

5'-TCGAGTCCTCAAGGACGAATTTCCCCAGC-3' (SEQUENCE ID. NO: 2)

3'-CAGGAGTTCCTGCTTAAAGGGGTCGAGCT-5' (SEQUENCE ID. NO: 3)

wherein the changed base is underlined and which, for purposes of theSequence Listing hereinbelow, consists of the following sequencesexpressed in the 5' to 3' direction:

5'-TCGAGTCCTCAAGGACGAATTTCCCCAGC-3' (SEQUENCE I.D. NO: 2)

and

5'-TCGAGCTGGGGAAATTCGTCCTTGAGGAC-3' (SEQUENCE I.D. NO: 3)

One fifth of the ligation mix was transformed into E. coli strain DH5-αand the transformants were plated on LB plates containing 100 μg/mlampicillin. DNA was prepared from 16 transformant colonies and 1-2 μgplasmid DNA was digested with EcoRI. DNA fragments were separatedelectrophoretically on 0.7% agarose and visualized with ethidiumbromide. Five micrograms of DNA from several plasmids, which released asingle 2.5 kb EcoRI DNA fragment rather than 1.6 and 0.9 kb DNAfragments, were then digested with SacI to release a 243 bp DNA fragmentcontaining the altered internal XhoI fragment. Fragments were separatedelectrophoretically on a 5% polyacrylamide gel and visualized byethidium bromide staining. The 243 bp SacI DNA fragment from one plasmidwas electroeluted and ligated with 200 ng of pBluescript+SK DNA whichhad been digested with SacI and treated with alkaline phosphatase. Theligation mixture was transformed into E. coli XL1-Blue cells (StratageneCloning Systems, La Jolla, Calif.) and the transformants were plated onLB plates containing 100 μg/ml ampicillin and IPTG and X-GAL asrecommended by the supplier for blue-white selection. Plasmid DNA wasprepared from 10 white transformant colonies, digested with SacI andfragments were separated electrophoretically on a 5% polyacrylamide gelto confirm the presence of the insert. DNA from two plasmids containingthe insert was sequenced using the dideoxy chain termination method(Sequenase kit, United States Biochemical Corp. Cleveland, Ohio) toconfirm the correct orientation of the XhoI DNA fragment within the LEU2coding sequence. One of the plasmids containing a LEU2 gene with analtered internal XhoI fragment was named pLD1049.

Approximately 500 ng of DNA from pNB257 was then digested with EcoRI tolinearize the plasmid, treated with alkaline phosphatase, and ligated toapproximately 200 ng of a 2.5 kb EcoRI DNA fragment containing the Y.lipolytica LEU2 gene isolated from pLD1049. One fifth of the resultingligation mix was transformed into E coli strain DH5-α, and thetransformants were plated on LB plates containing 100 μg/ml ampicillin.Plasmid DNA was prepared from a number of transformant colonies and 1-2μg of each plasmid DNA was digested with EcoRI. DNA fragments wereseparated electrophoretically on 0.7% agarose and visualized withethidium bromide. A transformant containing the 2.5 kb EcoRI DNAfragment containing the LEU2 gene was designated as pNB258. PlasmidpNB258 has been deposited in the form of an E. coli DH5α-transformant inthe American Type Culture Collection as described hereinbelow and hasbeen assigned deposit number ATCC 69353.

A series of deletions in the Y. lipolytica LEU2 gene promoter nucleotidesequence were prepared as follows. The below described deletionsgenerated from SEQUENCE I.D. NOS: 4 through 8 and 12 were spaced atabout 50 base pair intervals. Deletions generated from SEQUENCE I.D.NOS: 9, 10 and 11 made single base changes within the presumed "TATA"box of the LEU2 gene such that, for SEQUENCE I.D. NO. 9 the nucleotideat position 724 of the LEU2 gene was changed from A to G; for SEQUENCEI.D. NO. 10, the nucleotide at position 725 of the LEU2 gene was changedfrom T to G; and for SEQUENCE I.D. NO. 11, the nucleotide at position722 of the LEU2 gene was changed from A to G. Further, the primers weredesigned to provide appropriate restriction enzyme sites, e.g. BamHI andEcoRI, for subsequent use in subcloning of the sequences to begenerated. It will be appreciated by those skilled in the art enabled bythis disclosure that any deletion or series of deletions at any intervalor intervals can be prepared according to this invention as well as bythe use of other appropriate restriction sites.

The following 5' primers were synthesized using the Milligen CyclonePlus Synthesizer (Model 8400; Millipore Corp., Bedford, Mass.). Asdiscussed above, the primers were designed to result in nucleotidesequences having various 5' termini but also containing restrictionenzyme sites suitable for subsequent subcloning. Tabulated below are theprimers that were prepared.

                                      TABLE I                                     __________________________________________________________________________                                      5' Terminus                                                              Sequence                                                                           of Resulting                                Nucleotide Sequence          I.D. NO:                                                                           LEU 2 Gene                                  __________________________________________________________________________    GCAGGATCCG AATTCCTTGA CGATCTCGTA TGTC                                                                      4    559                                         GCAGGATCCG AATTCGCTGG GGTACGTTCG ATAG                                                                      5    599                                         GCAGGATCCG AATTCTAGCC GATACCGCAC TACC                                                                      6    648                                         GCAGGATCCG AATTCTCTTC CACATAGCAC GGGC                                                                      7    693                                         GCAGGATCCG AATTCCGTAT ATATACAAGA GCGTTTGCC                                                                 8    718                                         GCAGGATCCG AATTCCGTAT GTATACAAGA GCGTTTGCC                                                                 9    718                                         GCAGGATCCG AATTCCGTAT AGATACAAGA GCGTTTGCC                                                                 10   718                                         GCAGGATCCG AATTCCGTGT ATATACAAGA GCGTTTGCC                                                                 11   718                                         GCAGGATCCG AATTCCCACA GATTTTCACT CC                                                                        12   745                                         __________________________________________________________________________

Each of the primers listed in Table I, above, contains a restrictionenzyme recognition site for BamHI and EcoRI towards the 5' end thereof.

The Y. lipolytica LEU2 structural gene nucleotide sequence begins atnucleotide 799 of SEQUENCE I.D. NO: 1. A 3' primer was prepared and thesequence thereof was chosen to include a convenient restriction sitewithin the structural coding region of the LEU2 gene. The site chosenwas a StuI site which is located at nucleotide 919 of SEQUENCE I.D.NO: 1. Of course, other appropriate restriction enzyme sites could beused. The 3' primer used in the constructions discussed below containedthe nucleotide sequence CACAAACTCG GTGCCGGAGG CC (SEQUENCE I.D. NO: 13).The 3' primer was prepared according to the method described above forthe preparation of the 5' primers listed in Table I.

The method of polymerase chain reaction (PCR) was used to preparemultiple copies of nucleotide sequences having 5' termini correspondingto the 5' termini of the primers listed in Table I, above, and having 3'termini corresponding to the 3' terminus of the 3' primer of SEQUENCEI.D. NO: 13. The PCR was conducted using a Perkin-Elmer-Cetus PCRReagent Kit (Catalog N801-0055) in a Perkin-Elmer Cetus DNA ThermalCycler (Norwalk, Conn.). The reaction mixture contained 10 mM Tris-HCl,pH8.3, 50 mM KCl, 1.5 mM MgCl₂, 200 μM each of dATP, dCTP, dGTP anddTTP, 1 μM of the 5' primer, 1 μM of the 3' primer, 2.5 units ofampliTaq DNA polymerase and approximately 10 ng of pNB258 DNA in a totalvolume of 100 μL. The reaction was run for 30 cycles using the followingparameters: melt at 94° C. for 1.5 min., anneal at 50° C. for 2 min.,extend at 72° C. for 3 min.

Modified Y. lipolytica LEU2 genes with deleted promoters wereconstructed as follows and as depicted in FIG. 1. Each PCR generated DNAfragment, prepared as described immediately above, was digested withBamHI and StuI, and gel isolated. Plasmid pNB258 DNA was digested withBamHI and StuI to remove the wild-type LEU2 promoter and LEU2 codingsequences up to the StuI site which formed the 3' end of eachPCR-generated DNA fragment as well as a 0.68 kb StuI DNA fragmentinternal to the LEU2 gene. The 6.2 kb BamHI-StuI DNA vector fragment aswell as the 0.68 kb StuI internal DNA fragment were gel-isolated usingelectrophoresis. Approximately 200 ng of each PCR-generated fragment wasthen ligated in separate reactions to approximately 500 ng of the 6.2 kbBamHI-StuI DNA fragment and 200 ng of the 0.68 kb StuI DNA fragment.Approximately one-fifth of each ligation mix was transformed into E.coli DH5-α, and the transformants were plated on LB plates containing100 μg/ml ampicillin. Plasmid DNA was prepared from a number oftransformants and digested separately with EcoRI and StuI restrictionenzymes to identify those plasmids which contained the correct DNAfragments. Correct plasmids were sequenced by the dideoxy chaintermination method (TaqTrack Sequencing System, Promega, Madison Wis.)using as primer one or more of the following oligodeoxynucleotides(synthesized as described previously (SEQUENCE I.D. NOS: 5 and 14) orcommercially available (T3 primer; Stratagene Cloning Systems, La JollaCalif.)):

(1) 5'-CTCCTCCAATGAGTCGG-3' (SEQUENCE I.D. NO: 14); this primerhybridized to LEU2 gene sequences within the internal 0.68 kb StuI DNAfragment and was designed to generate DNA sequence information upstreamfrom the StuI site at the 3' end of the PCR DNA fragment;

(2) SEQUENCE I.D. NO: 5; this primer hybridized to LEU2 sequences 5' ofthe open reading frame and generated sequence information from the 3'end of the promoter region downstream through the LEU2 open readingframe; and

(3) A commercially available T3 primer which hybridized to vectorsequences upstream of the 5' end of the PCR-generated DNA fragment.

DNA sequence information thus generated was used to determine that theinternal StuI DNA fragment was oriented correctly and that eachPCR-generated DNA sequence was authentic.

2. CONSTRUCTION OF Y. lipolytica HOST STRAIN WITH DELETED LEU2 GENE(LEU2Δ).

Y. lipolytica host strains wherein the LEU2 sequences homologous to thesequences of the defective LEU2 genes of this invention are deleted wereconstructed. To construct such strains, the following plasmid (pNB243)was prepared. The construction of that plasmid is schematically depictedin FIG. 2. The 1.7 kb SalI fragment containing the URA3 gene from pLD106was converted to blunt ends by filling in with the large fragment ofKlenow polymerase and approximately 200 ng of the 1.7 kb SalI bluntedfragment was ligated to approximately 500 ng of EcoRI-digested, Klenowblunted Bluescript SK⁺ vector (Stratagene Cloning Systems, La Jolla,Calif.). A blunted URA3 gene can also be recovered from pNB258,described hereinabove, by, for example, digestion with KpnI and HindIIIand blunting with T4 DNA polymerase. Approximately one-fifth of theligation mix was transformed into E. coli strain DH5-α and thetransformants were plated onto LB plates containing 100 μg/mlampicillin. Plasmid DNA was prepared from a number of transformants anddigested with HindIII and BamHI restriction enzymes. Fragments wereseparated electrophoretically on a 1.0% agarose gel and visualized bystaining with ethidium bromide.

The correct plasmid contained a 1.7 kb DNA fragment containing URA3sequences and was named pNB241. Five hundred nanograms of pNB241 DNA wasthen digested with SalI, treated with alkaline phosphatase and ligatedtogether with 200 ng of a 5.3 kb SalI DNA fragment from pLD28 whichcontained LEU2 coding sequences along with DNA sequences flanking LEU2in the Y. lipolytica chromosome. Plasmid pLD28 is described in U.S. Pat.No. 5,071,764 and is described and claimed in U.S. Pat. No. 4,880,741.Approximately one-fifth of the ligation mix was transformed into E. colistrain DH5-α and the transformants were plated onto LB plates containing100 μg/ml ampicillin. DNA was prepared from a number of transformantsand digested with SalI. Fragments were separated electrophoretically ona 1.0% agarose gel and visualized by staining with ethidium bromide.Digestion of the correct plasmid released a 5.3 kb SalI DNA fragment.This plasmid was named pNB242. DNA from plasmid pNB242 was then digestedwith EcoRI to release 1.6 and 0.9 kb DNA fragments containing LEU2coding sequences. The remaining 7.4 kb linear vector waselectrophoretically gel isolated, ligated and the ligation mix used totransform E. coli strain DH5-α. Transformants were plated on LB platescontaining 100 μg/ml ampicillin. DNA was isolated from a number oftransformants, digested with SalI and fragments were separatedelectrophoretically on a 1.0% agarose gel. The correct plasmid (pNB243)contained a 2.8 kb SalI DNA fragment.

Plasmid pNB243 contains three XhoI restriction sites: two within theURA3 gene and one within the 2.8 kb SalI DNA fragment of LEU2. PlasmidpNB243 was partially digested with XhoI to linearize the vector asfollows. Approximately 2 μg of pNB243 DNA was treated respectively with10 units of XhoI enzyme for 5, 10 and 20 minutes at 37° C. The reactionwas stopped by ethanol precipitation of the reaction mixture and samplesof the digested DNA were analyzed electrophoretically on a 0.8% agarosegel. The five minute time point, which gave the highest proportion of7.4 kb linear vector, was chosen to use for transformation of Y.lipolytica strain NBL369 (MATB,bio6::BIO(pBR322), leu2-40, xpr2-1002,ura3Δ). Transformants were plated on minimal medium lacking uracil toselect for Ura+ transformants. DNA was isolated from a number of Ura+transformants and digested separately by SphI and SalI. The resultingDNA fragments were separated by agarose gel electrophoresis andtransferred to Hybond N nylon membrane (Amersham Corp., ArlingtonHeights, Ill.). The membranes were hybridized to a 2.4 kb EcoRI-SalI DNAfragment of LEU2 from pNB243. The disappearance of the parental 7.5 kbSphI LEU2 DNA fragment and the appearance of a new approximately 16 kbSphI DNA fragment indicated that the vector had integrated properly atthe LEU2 locus. The appearance of both the 5.3 kb SalI (parental) and2.9 kb SalI (deletion) LEU2 DNA fragments verified that both thewild-type and the deletion LEU2 genes were present. Two suchtransformants were named NBL461 and NBL462.

NBL461 and NBL462 were grown under non-selective conditions on rich YPDmedium for two days to allow for loss or "pop-out" of the URA3 marker byhomologous recombination. The cells were then plated onto 5-fluorouracilselection plates supplemented with leucine and uracil as described byBoeke, J. D., et al., Methods in Enzymology 154: 164-175 (1987). DNA wasprepared from several 5-fluorouracil (ura⁻) survivors, digested withSalI and screened by Southern hybridization analysis as described aboveusing the 0.4 kb SalI-EcoRI fragment of pNB243 as a probe. Two of thetransformants, NBL463 and NBL464, had lost the wild-type sized SalIfragment, and had retained only the leu2Δ-sized SalI fragment.

The strains listed below were deposited on Jul. 22, 1993, under theterms of the Budapest Treaty in the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md. 20852, United States of America, arecognized depository affording permanence of the deposits and readyaccessibility thereto by the public if a patent is granted on thisapplication. The deposits are available during the pendency of thisapplication to one determined by the Commissioner of the United StatesPatent and Trademark Office to be entitled thereto under 37 CFR 1.14 and35 U.S.C. 122, and in accordance with foreign patent laws in countrieswherein counterparts of this application, or its progeny, are filed. Allrestrictions on the availability to the public of the microorganismsdeposited will be irrevocably removed upon granting of the patent.

    ______________________________________                                        STRAIN          ATCC Deposit Number                                           ______________________________________                                        Y. lipolytica NBL464                                                                          ATCC 74234                                                    E. coli DH5-α/pNB258                                                                    ATCC 69353                                                    E. coli DH5-α/pNB650                                                                    ATCC 69354                                                    E. coli DH5-α/pNB268                                                                    ATCC 69355                                                    ______________________________________                                    

3. EVALUATION OF DEFECTIVE LEU2 GENES.

To evaluate the LEU2 gene promoter deletion plasmids which were preparedas described above, Y. lipolytica leu 2Δ strain NBL464(ATCC 74234) wastransformed separately by pNB258 and each of the LEU2 promoter deletionplasmids. Each plasmid was digested with PstI prior to transformation inorder to target integration by homologous recombination to the ura3Δregion of the Y. lipolytica chromosome. The transformants were selectedon complete minimal medium without uracil and the Ura⁺ transformantswere then screened for the Leu phenotype by their ability to grow oncomplete minimal medium lacking leucine. Tabulated below are the resultsof that screen.

                  TABLE II                                                        ______________________________________                                                   Growth on Complete Minimal                                         Plasmid    Medium minus Leucine                                               ______________________________________                                        pNB258     Good                                                               pNB271     Good                                                               pNB272     Good                                                               pNB276     Weak                                                               ______________________________________                                    

Plasmid pNB276 contains a weakly-complementary LEU2 gene and, hence,contains a defective LEU2 gene promoter having its 5' terminus atposition 693 of the LEU2 gene. Other LEU2 promoter deletion genesproduced according to this invention and contained in plasmidsdesignated pNB656 (5' terminus at position 718 of the LEU2 gene), pNB652(5' terminus at position 718 of the LEU2 gene and containing nucleotideG instead of A at position 724), pNB653 (5' terminus at position 718 ofthe LEU2 gene and containing nucleotide G instead of T at position 725),pNB654 (5' terminus at position 718 of the LEU2 gene and containingnucleotide G instead of A at position 722) and pNB313 (5' terminus atposition 745 of the LEU2 gene) provided similar results.

4. CONSTRUCTION OF MULTIPLE-INTEGRATION VECTOR COMPRISING rDNASEQUENCES.

Construction of a multiple integration vector (pNB308) is schematicallydepicted in FIG. 3. Plasmid pNB276, prepared and identified as describedabove, was modified by deleting the SaclI site within the polycloningregion of the plasmid. The deletion was accomplished by digesting pNB276with SaclI. Then, the SaclI digested pNB276 was made blunt-ended withKlenow fragment and then blunt-end ligated. The blunt-end ligatedplasmids were used to transform E. coli DH5-α and the correct construct(pNB305) was identified by restriction analysis. Ribosomal DNA (rDNA)sequences were isolated from pNB650 by digesting plasmid DNA withHindIII. pNB650 contains a 2.8 kb NcoI fragment of rDNA (Clare, J. J.,et al., Curr Genet 10:449-452 (1986)), in which the NcoI sites have beenconverted to HindIII sites by blunting with Klenow fragment and ligatingto HindIII linkers followed by ligation to HindIII-digestedpBluescript+SK (Stratagene Cloning Systems, La Jolla, Calif.). PlasmidpNB650 has been deposited with the American Type Culture Collection inthe form of an E. Coli DH5-α transformant and has been assigned depositnumber ATCC 69354 as described above. The 2.8 kb HindIII rDNA containingfragment from pNB650 was isolated by standard agarose gelelectrophoresis methods. The isolated 2.8 kb HindIII rDNA fragment wasthen ligated to HindIII digested pNB305. The resulting ligation mixturewas used to transform E. coli DH5-α. DNA from a number of transformantswas digested by HindIII and fragments separated electrophoretically on a0.7% agarose gel. The correct plasmid contained a 2.8 kb HindIII DNAfragment and was named pNB308.

5. CONSTRUCTION OF MULTIPLE INTEGRATION VECTOR CONTAINING STRAINS OF Y.lipolytica.

Plasmid pNB308, prepared as described above, was digested with SaclIwhich cuts one time within the rDNA sequence in the plasmid. The linearplasmid was thus targeted to integrate by homologous recombination withthe repeated rDNA sequences of the host Y. lipolytica strain. Strain Y.lipolytica NBL464 (ATCC 74234) was transformed with SaclI digestedpNB308 according to the standard procedure described above.Transformants were selected on complete minimal medium without leucine.There appeared Leu⁺ transformants of large and small colony type.

DNA was isolated from large transformant colonies, digested with SalI,run on an agarose gel, transferred to Hybond N membranes, and probedwith a 0.49 kb BstXI-SalI fragment of URA3 DNA which would hybridizeboth to the single copy chromosomal ura3Δ gene (on a 1.0 kb SalIfragment) as well as to each copy of URA3 contributed by pNB308 (on a3.5 kb SalI fragment). Hybridization signals were quantitated using aBeta-Scope 603 Blot Analyzer (Betagen, Waltham, Mass.), and plasmid copynumber for each transformant was determined by calculating the folddifference in total counts for the 3.5 kb band compared with the 1.0 kbband. Summarized in Table III, below, are the copy numbers for thetransformants studied by Southern analysis.

                  TABLE III                                                       ______________________________________                                        Transformant  MIV Copy Number                                                 ______________________________________                                         1            3                                                                2            3                                                                3            2                                                                4            2                                                                5            4                                                                6            5                                                                7            6                                                                8            12                                                               9            11                                                              10            8                                                               11            5                                                               12            5                                                               13            6                                                               14            4                                                               15            4                                                               16            3                                                               17            5                                                               18            11                                                              19            6                                                               20            4                                                               ______________________________________                                    

To confirm that the vector was integrated within rDNA sequences,transformant DNA was digested with SaclI, which cuts once within theplasmid within rDNA sequences, run on an agarose gel, transferred toHybond N membranes, and probed with a 0.49 kb BstXI-SalI DNA fragmentfrom URA3. A 10.8 kb band, the size of the intact plasmid, was detected,which confirmed that the vector had integrated within rDNA sequences andwithout major rearrangement.

The stability of transformants 8 and 9 was studied. Transformants 8 and9 were inoculated 1:100 into separate YPD medium, grown 24 hours at 28°C., diluted 1:100 into fresh YPD medium and regrown for a total of threecycles. Cells were sampled at 24, 48 and 72 hours (corresponding toapproximately 12, 24 and 36 generations). DNA was prepared from the cellsamples and analyzed for copy number as described above. The copynumbers of transformants 8 and 9 did not change during continued growthin rich, non-selective medium.

7. CONSTRUCTION OF PROINSULIN ANALOG MULTIPLE INTEGRATIVE EXPRESSIONVECTOR.

The starting plasmid in the construction of a proinsulin analog multipleintegrative expression vector was plasmid pXPRURAcas, which contains theXPR2 promoter, pre- and pro-sequences followed by a multiple cloningsite, and XPR2 terminator sequences along with a wild-type URA3 gene asselectable marker. The construction of a proinsulin analog multipleintegrative expression vector is shown in FIG. 4. Plasmid pXPRURAcas wasdigested with KpnI, blunted by treatment with T4 polymerase, andredigested with SaclI. This vector fragment was ligated to a proinsulinanalog (A14trp) coding sequence provided by Scios Nova, Inc.,Mountainview, Calif. which had the following sequence along with5'-blunt and 3'-SaclI restriction ends: ##STR1##

For purposes of the Sequence Listing hereinbelow, that sequence isrepresented as SEQUENCE I.D. NO: 14 for the 5'-3' single strand andSEQUENCE I.D. NO: 15 for the 3'-5' single strand, though presented asthe corresponding 5'-3' strand in SEQUENCE I.D. NO: 15.

This ligation created an in-frame fusion between the last codon of theY. lipolytica XPR2 pro region and the first codon of proinsulin analogA14trp. Part of the ligation mix was transformed into E. coli DH5-α, andthe transformants were plated on LB containing 100 μg/ml ampicillin.Plasmid DNA was prepared from a number of transformants, digested withHind III, and the fragments separated electrophoretically on 0.8%agarose gels. The correct construction showed release of a 2.8 kbHindIII DNA fragment due to introduction of an additional HindIII sitewithin the proinsulin analog gene fragment. Several positive clones wereidentified and sequenced to ensure the fidelity of the junction sequencebetween the XPR2 pro region and the proinsulin gene, as well as thefidelity of the PCR-generated proinsulin analog gene itself. Plasmid DNAwas sequenced using a TaqTrack kit and deaza-nucleotides (Promega,Madison, Wis.) plus a primer that hybridized within the pro region ofXPR2 to generate sequence information across the XPR2 pro-proinsulinjunction and through the proinsulin gene. The sequence of this primer isTACACGGATGGATCTGG (SEQUENCE I.D. NO: 16). One such proinsulin analogexpression vector was named pXPRURAINS.

Plasmid pXPRURAINS was then digested with MluI (a unique site within theXPR2 promoter) and SacI (a unique site at the 3' end of the proinsulinanalog gene), and a 1.7 kb DNA fragment was electrophoretically gelisolated. The 1.7 kb DNA fragment was ligated to a 7.3 kb DNA fragmentgenerated by digesting pXPRLEUcas with MluI and SaclI. PlasmidpXPRLEUcas, also designated pNB268, has been deposited in the AmericanType Tissue Collection as described hereinabove and has been assigneddeposit number ATCC 69355. The ligation mix was transformed into E. coliDH5-α and transformants were plated on LB containing 100 μg/mlampicillin. DNA was prepared from several transformants and digestedwith HindIII to identify a correct plasmid (named pXPRLEUINS) asdepicted in FIG. 4.

Plasmid pXPRLEUINS was then digested with SaclI, treated with Klenowfragment to generate blunt ends and religated. This destroyed the SaclIsite at the 3' end of the proinsulin analog gene so that the SaclI sitewithin the rDNA fragment of pNB650 would be unique, and digestion atthis site could be used to target integration of the multipleintegrative expression vector to rDNA loci. The ligation mix wastransformed into E. coli DH5-α and transformants were plated on LBcontaining 100 μ/ml ampicillin. Transformant DNA was prepared anddigested with SaclI. A correct plasmid was identified which no longerwas linearized upon digestion with SaclI. That plasmid was namedpXPRLEUINS(-SaclI). Plasmid PXPRLEUINS(-SaclI) was then digested withEcoRI to remove the wild-type LEU2 gene, and the 7.4 kb vector fragmentwas ligated to a 2.2 kb EcoRI DNA fragment containing the defective LEU2((d) LEU) gene isolated from pNB276. The ligation mix was transformedinto E. coli DH5-α, and transformants were plated on LB containing 100μg/ml ampicillin. The correct plasmid was identified by restrictiondigestion with EcoRI, which released a single 2.2 kb DNA fragmentcontaining the (d)LEU gene instead of the wild-type 1.6 and 0.9 kb EcoRIDNA fragments. The correct plasmid was designated pXPR(d)LEUINS. Thisplasmid was then linearized by partial digestion with HindIII, asdescribed hereinabove, dephosphorylated by treatment with calfintestinal phosphatase and ligated to the 2.8 kb HindIII DNA fragmentcontaining rDNA sequences from pNB650 described above. The ligation mixwas transformed into E. coli DH5-α and transformants were plated on LBcontaining 100 μg/ml ampicillin. The correct plasmid, in which rDNAsequences had been ligated at the HindIII site upstream of the XPR2promoter, was identified by restriction digestion with EcoRI, whichreleased fragments of 4.9, 5.4, and 2.4 kb. This plasmid was designatedpMIVINS.

8. CONSTRUCTION OF PROINSULIN ANALOG EXPRESSING MULTIPLE INTEGRATIONVECTOR TRANSFORMANTS OF Y. lipolytica.

Plasmid pMIVINS was digested with SaclI to target it to the rDNA locusand transformed into Y. lipolytica strain NBL464 (ATCC 74234).Transformants were obtained after 48 hours at 29° C. on complete minimalmedium minus leucine. DNA was obtained from two sets of transformants inseparate experiments designated 1 and 2 in Table IV below. The genomicDNA was digested with HindIII and EcoRI (experiment 1) or with HindIII(experiment 2), fragments were separated electrophoretically on an 0.7%agarose gel and blotted to Hybond N membrane. The membrane was probedwith a labeled 0.8 kb PstI-MluI DNA fragment isolated from the promoterregion of XPR2. This probe hybridized to a 3.7 kb HindIII DNA fragmentfrom the genomic copy of XPR2 (from either the HindIII-EcoRI digest orthe HindIII digest) as well as to a 2.8 kb HindIII DNA fragment frompMIVINS (from either the HindIII-EcoRI digest or the HindIII digest).The blots were scanned using a BetaScope 603 Blot Analyzer as previouslydescribed to determine the number of copies of pMIVINS integrated intoeach transformant strain. Control transformants (NBL449 and NBL451), notnecessary for practice of this invention and which were prepared asdescribed below, were also analyzed by the same procedure.

Control transformants NBL449 and NBL451 were prepared as follows.Plasmid pXPRURAINS was digested with XhoI to target integration thereofinto the URA3 locus, and transformed into Y. lipolytica strain NBL369(MATB, bio-6::BIO(pBR322),leu2-40, xpr2-1002, ura 3Δ). Transformantswere plated on complete minimal medium uracil to identify Ura⁺transformants. Then, pXPRLEUINS was digested with XhoI to targetintegration thereof into the LEU2 locus, and used to separatelytransform Y. lipolytica strain NBL369 and a Ura⁺ transformant obtainedas described immediately above. Transformants from each transformationwere plated on complete minimal medium minus leucine or minus uracil andleucine to identify Leu⁺ and Leu⁺ Ura⁺ transformants, respectively. ALeu⁺ transformant containing one copy of pXPRLEUINS was named NBL449. ALeu⁺ Ura⁺ transformant containing one copy of pXPRURAINS and one copy ofpXPRLEUINS (i.e., two copies of the proinsulin analog coding sequence)was named NBL451. Table IV, below, presents the copy number of thetransformant strains so tested as well control transformants NBL449 andNBL451.

                  TABLE IV                                                        ______________________________________                                        Transformant   Copy Number                                                    ______________________________________                                        1-1            5                                                              1-2            3                                                              1-3            4                                                              1-4            4                                                              1-5            5                                                              1-6            4                                                              1-7            3                                                              1-8            3                                                              1-9            3                                                              NBL449         1                                                              NBL451         2                                                              2-1            4                                                              2-2            4                                                              2-3            5                                                              2-4            5                                                              2-5            3                                                              2-6            4                                                              2-7            2                                                              2-8            2                                                              2-9            5                                                               2-10          4                                                               2-11          2                                                               2-12          4                                                               2-13          4                                                               2-14          5                                                               2-15          2                                                               2-16          5                                                              NBL449         1                                                              NBL451         2                                                              ______________________________________                                    

Radioimmunoassay (RIA) was used to determine the relative amount ofproinsulin analog related material secreted by the Y. lipolyticatransformants comprising multiple copies of pMIVINS. The above-describedtransformant strains were grown in Medium A (5% Bacto-peptone, 1%glucose, 0.1% yeast extract) for 48 hours and then the supernatants werecollected. Then, C-peptide RIAs, which measure free C-peptide ofinsulin, were conducted using a commercially available kit (IncstarCorp., Stillwater, N. Mex.). The C-peptide antibody reactive materialwas found to increase with increasing copy number. The five copytransformant secreted about 4 to 4.5 times the amount of C-peptiderelated protein as did the single copy proinsulin expression strain.

9. CONSTRUCTION OF INSULINOTROPIN MULTIPLE INTEGRATIVE EXPRESSION VECTOR

A synthetic gene encoding insulinotropin was prepared according to thefollowing scheme: SEQUENCE I.D. NOS. 17, 18, 19 and 20 having thefollowing sequences:

CACGCCGAGGGCACCTTCACCTCCGACGTCTCCTC (SEQUENCE I.D. NO: 17);

CCGGGTGCGGCTCCCGTGGAAGTGGAGGCTGCAGAGGAGGATGG (SEQUENCE I.D. NO: 18);

CTACCTGGAGGGACAGGCCGCCAAGGAGTTCATCGCCTGGCTGGTCAAGG GACGAGGATAGT(SEQUENCE I.D. NO: 19); and

ACCTCCCTGTCCGGCGGTTCCTCAAGTAGCGGACCGACCAGTTCC CTGCTCCTATCAGATC (SEQUENCEI.D. NO: 20)

were obtained from Genosys Biotechnologies, Inc., The Woodlands, Tex.Then, SEQUENCE I.D. NOS: 17 and 18 were hybridized together by heatingat 100° C. for 10 min. and cooling slowly to room temperature andSEQUENCE I.D. NOS: 19 and 20 were hybridized together under the sameconditions. The resulting hybridized sequences were treated withpolynucleotide kinase and ligated using T4 DNA ligase to yield a 100 bpfragment encoding insulinotropin and containing an ApaI sticky 5' endand an XbaI sticky 3' end. The fragment was then ligated into ApaI/XbaIdigested plasmid pBluescript+KS (Stratagene Cloning Systems, LaJolla,Calif.) for use in sequence verification. A plasmid containing theverified coding sequence was designated pNB716. Once the sequence wasverified, an initial expression vector (pNB747) was constructed asfollows and as depicted schematically in FIG. 5.

Plasmid pNB268 (ATCC 69355) DNA was digested with KpnI and the stickyends were blunted using T4 DNA polymerase. The DNA was then digestedwith XbaI to receive the 3' end of the insulinotropin encoding fragmentprepared as described above. The DNA of plasmid pNB716 containing theinsulinotropin encoding sequence was digested with ApaI and the stickyends were blunted with T4 DNA polymerase. The DNA was then digested withXbaI. The resulting 100 bp fragment was electrophoretically isolated on2.0%, NuSieve agarose (FMC BioProducts, Rockland, Me.) gel and ligatedto the KpnI digested, blunted and XbaI digested DNA of pNB268. Theresulting ligation mix was used to transform E. coli DH5-α. Plasmidshaving the correct construction were identified by restriction enzymeanalysis and the blunted junction region was sequenced to verity anin-frame fusion between the XPR2 prepro sequences and the insulinotropinencoding sequence. One such plasmid, pNB747, is shown in FIG. 5.

Plasmid pNB747 was digested with EcoRI to remove the wild-type LEU2gene, and the 7.4 kb vector fragment was ligated to a 2.2 kb EcoRI DNAfragment containing the defective LEU2 ((d)LEU) gene isolated frompNB276. The ligation mix was transformed into E. coli DH5-α andtransformants were plated on LB containing 100 μg/ml ampicillin. Thecorrect plasmid was identified by restriction digestion with EcoRI,which released a single 2.2 kb DNA fragment containing the (d) LEU geneinstead of the wild-type 1.6 and 0.9 kb EcoRI DNA fragments. The correctplasmid was designated pNB751.

Plasmid pNB751 was then digested with SaclI, treated with Klenowfragment to generate blunt ends and religated. This destroyed the SaclIsite at the 3' end of the insulinotropin gene so that the SaclI sitewithin the rDNA fragment of pNB650 would be unique, and digestion atthis site could be used to target integration of the multipleintegrative expression vector to rDNA loci. The ligation mix wastransformed into E. coli DH5-α and transformants were plated on LBcontaining 100 μ/ml ampicillin. Transformant DNA was prepared anddigested with SaclI. A correct plasmid was identified which no longerwas linearized upon digestion with SaclI. That plasmid was namedpXPRLEUIST(-SaclI). Plasmid pXPRLEUIST(-SaclI) was then digested withEcoRI to remove the wild-type LEU2 gene, and the 7.4 kb vector fragmentwas ligated to a 2.2 kb EcoRI DNA fragment containing the defective LEU2((d) LEU) gene isolated from pNB276. The ligation mix was transformedinto E. coli DH5-α, and transformants were plated on LB containing 100μg/ml ampicillin. The correct plasmid was identified by restrictiondigestion with EcoRI, which released a single 2.2 kb DNA fragmentcontaining the (d)LEU gene instead of the wild-type 1.6 and 0.9 kb EcoRIDNA fragments. The correct plasmid was designated pXPR(d)LEUIST. Thisplasmid was then linearized by partial digestion with HindIII, asdescribed hereinabove, dephosphorylated by treatment with calfintestinal phosphatase and ligated to the 2.8 kb HindIII DNA fragmentcontaining rDNA sequences from pNB650 described above. The ligation mixwas transformed into E. coli DH5-α and transformants were plated on LBcontaining 100 μg/ml ampicillin. The correct plasmid, in which rDNAsequences had been ligated at the HindIII site upstream of the XPR2promoter, was identified by restriction digestion with EcoRI, whichreleased fragments of 4.9, 5.4, and 2.4 kb. This plasmid was designatedpMIVIST.

10. CONSTRUCTION OF INSULINOTROPIN EXPRESSING MULTIPLE INTEGRATIONVECTOR TRANSFORMANTS OF Y. lipolytica.

Plasmid pMIVIST was used to transform Y. lipolytica strain NBL464 (ATCC74234) after having been digested with SaclI. The resultingtransformants obtained on complete minimal medium minus leucinecontained multiple integrated vectors. Correlation between integratedcopy number and the level of expression of insulinotropin was unclear.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 20                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2810 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTCGACACCATATCATATAAAACTAACAATGCATTGCTTATTACGAAGACTACCCGTTGC60                TATCTCCACACCGTTATCTCCACGGTCCAAAGGCTGCTCAATGTGCTGCATACGTAACGT120               GGGGTGCAACCTTGAGCACATAGTACTTTTCCGAAAACCGGCGATAATTAAGTGTGCACT180               CCAACTTTTCACACTGAGCGTAAAATGTGGAGAAGAAATCGGCACTAAAAAGTCAGGTAG240               ACTGGAAAATGCGCCATGAAATGAATATCTCTTGCTACAGTAATGCCCAGCATCGAGGGG300               TATTGTGTCACCAACACTATAGTGGCAGCTGAAGCGCTCGTGATTGTAGTATGAGTCTTT360               ATTGGTGATGGGAAGAGTTCACTCAATATTCTCGTTACTGCCAAAACACCACGGTAATCG420               GCCAGACACCATGGATGTAGATCACCAAGCCTGTGAATGTTATTCGAGCTAAAATGCACA480               TGGTTGGTGAAAGGAGTAGTTGCTGTCGAATTCCGTCGTCGCCTGAGTCATCATTTATTT540               ACCAGTTGGCCACAAACCCTTGACGATCTCGTATGTCCCCTCCGACATACTCCCGGCCGG600               CTGGGGTACGTTCGATAGCGCTATCGGCATCGACAAGGTTTGGGTCCCTAGCCGATACCG660               CACTACCTGAGTCACAATCTTCGGAGGTTTAGTCTTCCACATAGCACGGGCAAAAGTGCG720               TATATATACAAGAGCGTTTGCCAGCCACAGATTTTCACTCCACACACCACATCACACATA780               CAACCACACACATCCACAATGGAACCCGAAACTAAGAAGACCAAGACTGACTCCAAGAAG840               ATTGTTCTTCTCGGCGGCGACTTCTGTGGCCCCGAGGTGATTGCCGAGGCCGTCAAGGTG900               CTCAAGTCTGTTGCTGAGGCCTCCGGCACCGAGTTTGTGTTTGAGGACCGACTCATTGGA960               GGAGCTGCCATTGAGAAGGAGGGCGAGCCCATCACCGACGCTACTCTCGACATCTGCCGA1020              AAGGCTGACTCTATTATGCTCGGTGCTGTCGGAGGCGCTGCCAACACCGTATGGACCACT1080              CCCGACGGACGAACCGACGTGCGACCCGAGCAGGGTCTCCTCAAGCTGCGAAAGGACCTG1140              AACCTGTACGCCAACCTGCGACCCTGCCAGCTGCTGTCGCCCAAGCTCGCCGATCTCTCC1200              CCCATCCGAAACGTTGAGGGCACCGACTTCATCATTGTCCGAGAGCTCGTCGGAGGTATC1260              TACTTTGGAGAGCGAAAGGAGGATGACGGATCTGGCGTCGCTTCCGACACCGAGACCTAC1320              TCCGTTCCTGAGGTTGAGCGAATTGCCCGAATGGCCGCCTTCCTGGCCCTTCAGCACAAC1380              CCCCCTCTTCCCGTGTGGTCTCTTGACAAGGCCAACGTGCTGGCCTCCTCTCGACTTTGG1440              CGAAAGACTGTCACTCGAGTCCTCAAGGACGAATTCCCCCAGCTCGAGCTCAACCACCAG1500              CTGATCGACTCGGCCGCCATGATCCTCATCAAGCAGCCCTCCAAGATGAATGGTATCATC1560              ATCACCACCAACATGTTTGGCGATATCATCTCCGACGAGGCCTCCGTCATCCCCGGTTCT1620              CTGGGTCTGCTGCCCTCCGCCTCTCTGGCTTCTCTGCCCGACACCAACGAGGCGTTCGGT1680              CTGTACGAGCCCTGTCACGGATCTGCCCCCGATCTCGGCAAGCAGAAGGTCAACCCCATT1740              GCCACCATTCTGTCTGCCGCCATGATGCTCAAGTTCTCTCTTAACATGAAGCCCGCCGGT1800              GACGCTGTTGAGGCTGCCGTCAAGGAGTCCGTCGAGGCTGGTATCACTACCGCCGATATC1860              GGAGGCTCTTCCTCCACCTCCGAGGTCGGAGACTTGTTGCCAACAAGGTCAAGGAGCTGC1920              TCAAGAAGGAGTAAGTCGTTTCTACGACGCATTGATGGAAGGAGCAAACTGACGCGCCTG1980              CGGGTTGGTCTACCGGCAGGGTCCGCTAGTGTATAAGACTCTATAAAAAGGGCCCTGCCC2040              TGCTAATGAAATGATGATTTATAATTTACCGGTGTAGCAACCTTGACTAGAAGAAGCAGA2100              TTGGGTGTGTTTGTAGTGGAGGACAGTGGTACGTTTTGGAAACAGTCTTCTTGAAAGTGT2160              CTTGTCTACAGTATATTCACTCATAACCTCAATAGCCAAGGGTGTAGTCGGTTTATTAAA2220              GGAAGGGAGTTGTGGCTGATGTGGATAGATATCTTTAAGCTGGCGACTGCACCCAACGAG2280              TGTGGTGGTAGCTTGTTACTGTATATTCGGTAAGATATATTTTGTGGGGTTTTAGTGGTG2340              TTTGGTAGGTTAGTGCTTGGTATATGAGTTGTAGGCATGACAATTTGGAAAGGGGTGGAC2400              TTTGGGAATATTGTGGGATTTCAATACCTTAGTTTGTACAGGGTAATTGTTACAAATGAT2460              ACAAAGAACTGTATTTCTTTTCATTTGTTTTAATTGGTTGTATATCAAGTCCGTTAGACG2520              AGCTCAGTGCCATGGCTTTTGGCACTGTATTTCATTTTTAGAGGTACACTACATTCAGTG2580              AGGTATGGTAAGGTTGAGGGCATAATGAAGGCACCTTGTACTGACAGTCACAGACCTCTC2640              ACCGAGAATTTTATGAGATATACTCGGGTTCATTTTAGGCTCCGATTCGATTCAAATTAT2700              TACTGTCGAAATCGGTTGAGCATCCGTTGATTTCCGAACAGATCTCGGCAGTCTCTCGGA2760              TGTAGAATTAGGTTTCCTTGAGGCGAAGATCGGTTTGTGTGACATGAATT2810                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TCGAGTCCTCAAGGACGAATTTCCCCAGC29                                               (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TCGAGCTGGGGAAATTCGTCCTTGAGGAC29                                               (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GCAGGATCCGAATTCCTTGACGATCTCGTATGTC34                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCAGGATCCGAATTCGCTGGGGTACGTTCGATAG34                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GCAGGATCCGAATTCTAGCCGATACCGCACTACC34                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GCAGGATCCGAATTCTCTTCCACATAGCACGGGC34                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GCAGGATCCGAATTCCGTATATATACAAGAGCGTTTGCC39                                     (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GCAGGATCCGAATTCCGTATGTATACAAGAGCGTTTGCC39                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GCAGGATCCGAATTCCGTATAGATACAAGAGCGTTTGCC39                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GCAGGATCCGAATTCCGTGTATATACAAGAGCGTTTGCC39                                     (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GCAGGATCCGAATTCCCACAGATTTTCACTCC32                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CACAAACTCGGTGCCGGAGGCC22                                                      (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 279 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ATTCTTTGTGAACCAACACCTGTGCGGATCCCACCTGGTGGAAGCTCTCCACCTAGTGTG60                CGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGAGGGCAGAGGACCTGCAGGT120               GGGGCAGGTGGAGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTGGCCCTGGA180               GGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACCAGCATCTGCTCCCTCTA240               CCAGCTGGAGAACTACTGCAACTAGAAGCTTGGCCGCGG279                                    (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 271 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GGCCAAGCTTCTAGTTGCAGTAGTTCTCCAGCTGGTAGAGGGAGCAGATGCTGGTACAGC60                ATTGTTCCACAATGCCACGCTTCTGCAGGGACCCCTCCAGGGCCAAGGGCTGCAGGCTGC120               CTGCACCAGGGCCCCCGCCCAGCTCCACCTGCCCCACCTGCAGGTCCTCTGCCCTCCGGC180               GGGTCTTGGGTGTGTAGAAGAAGCCTCGTTCCCCGCACACTAGGTGGAGAGCTTCCACCA240               GGTGGGATCCGCACAGGTGTTGGTTCACAAA271                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      TACACGGATGGATCTGG17                                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CACGCCGAGGGCACCTTCACCTCCGACGTCTCCTC35                                         (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CCGGGTGCGGCTCCCGTGGAAGTGGAGGCTGCAGAGGAGGATGG44                                (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 62 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CTACCTGGAGGGACAGGCCGCCAAGGAGTTCATCGCCTGGCTGGTCAAGGGACGAGGATA60                GT62                                                                          (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 61 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      ACCTCCCTGTCCGGCGGTTCCTCAAGTAGCGGACCGACCAGTTCCCTGCTCCTATCAGAT60                C61                                                                           __________________________________________________________________________

What is claimed is:
 1. A modified Y. lipolytica LEU2 non-wild type genepromoter selected from the group consisting of nucleotides 693 to 798 ofSEQUENCE I.D. NO: 1, nucleotides 718 to 798 of SEQUENCE I.D. NO: 1,nucleotides 718 to 798 of SEQUENCE I.D. NO: 1 wherein nucleotide 724 ischanged from A to G, nucleotides 718 to 798 of SEQUENCE I.D. NO: 1wherein nucleotide 725 is changed from T to G, nucleotides 718 to 798 ofSEQUENCE I.D. NO: 1 wherein nucleotide 722 is changed from A to G,nucleotides 745 to 798 of SEQUENCE I.D. NO:
 1. 2. A modified Y.lipolytica LEU2 gene comprising the modified LEU2 gene promoteraccording to claim 1 functionally linked to a Y. lipolytica LEU2structural gene coding sequence.
 3. A vector comprising the modified Y.lipolytica LEU2 gene according to claim
 2. 4. The vector according toclaim 3 comprising a Y. lipolytica DNA sequence sufficient forintegrative transformation of Y. lipolytica at a locus other than theLEU2 locus of Y. lipolytica.
 5. The vector according to claim 4 whereinthe Y. lipolytica DNA sequence sufficient for integrative transformationis a Y. lipolytica ribosomal DNA sequence.
 6. An expression vectorcomprising the vector according to claim 5, a nucleotide sequence codingfor a polypeptide and a promoter functional in Y. lipolytica operablylinked to the nucleotide sequence coding for the polypeptide.
 7. A Y.lipolytica transformant comprising the expression vector according toclaim
 6. 8. A method of producing a polypeptide which comprisesfermenting the Y. lipolytica transformant according to claim 7 in anaqueous nutrient medium comprising assimilable sources of carbon,nitrogen and inorganic salts.
 9. The expression vector according toclaim 6 wherein the promoter functional in Y. lipolytica is the XPR2promoter of Y. lipolytica.
 10. The expression vector according to claim9 which comprises the signal, pro1- or pro2-sequence of the XPR2 gene ofY. lipolytica, or a functional fragment or equivalent thereof, operablylinked to the nucleotide sequence coding for the polypeptide.
 11. Theexpression vector according to claim 10 wherein the polypeptide is aheterologous polypeptide.
 12. The expression vector according to claim11 wherein the heterologous polypeptide is prochymosin, proinsulin,insulinotropin or human TGF-β3.
 13. A Y. lipolytica transformantcomprising the expression vector according to claim
 12. 14. A method ofproducing a polypeptide which comprises fermenting the Y. lipolyticatransformant according to claim 13 in an aqueous nutrient mediumcomprising assimilable sources of carbon, nitrogen and inorganic salts.15. A process of producing insulinotropin which comprises fermenting theY. lipolytica transformant according to claim 13 in an aqueous nutrientmedium comprising assimilable sources of carbon, nitrogen and inorganicsalts.
 16. A Y. lipolytica transformant comprising the expression vectoraccording to claim
 10. 17. A method of producing a polypeptide whichcomprises fermenting the Y. lipolytica transformant according to claim16 in an aqueous nutrient medium comprising assimilable sources ofcarbon, nitrogen and inorganic salts.
 18. A Y. lipolytica transformantcomprising the expression vector according to claim
 9. 19. A method ofproducing a polypeptide which comprises fermenting the Y. lipolyticatransformant according to claim 18 in an aqueous nutrient mediumcomprising assimilable sources of carbon, nitrogen and inorganic salts.20. An expression vector comprising the vector according to claim 4, anucleotide sequence coding for a polypeptide and a promoter functionalin Y. lipolytica operably linked to the nucleotide sequence coding forthe polypeptide.
 21. The expression vector according to claim 20 whereinthe promoter functional in Y. lipolytica is the XPR2 promoter of Y.lipolytica.
 22. The expression vector according to claim 10 whichcomprises the signal, pro1- or pro2-sequence of the XPR2 gene of Y.lipolytica, or a functional fragment or equivalent thereof, operablylinked to the nucleotide sequence coding for the polypeptide.
 23. Theexpression vector according to claim 22 wherein the polypeptide is aheterologous polypeptide.
 24. The expression vector according to claim23 wherein the heterologous polypeptide is prochymosin, proinsulin,insulinotropin or human TGF-β3.
 25. A Y. lipolytica transformantcomprising the expression vector according to claim
 24. 26. A method ofproducing a polypeptide which comprises fermenting the Y. lipolyticatransformant according to claim 25 in an aqueous nutrient mediumcomprising assimilable sources of carbon, nitrogen and inorganic salts.27. A Y. lipolytica transformant comprising the expression vectoraccording to claim
 20. 28. A method of producing a polypeptide whichcomprises fermenting the Y. lipolytica transformant according to claim27 in an aqueous nutrient medium comprising assimilable sources ofcarbon, nitrogen and inorganic salts.
 29. A method of producing a Y.lipolytica transformant comprising multiple integrated expressionvectors which method comprises:(a) transforming a Y. lipolytica strainhaving a deletion of the LEU2 gene thereof with the expression vectoraccording to claim 20 which has been linearized by cleaving theexpression vector in the DNA sequence sufficient for integrativetransformation; and (b) selecting for the best growing transformants ona medium which lacks leucine.
 30. A Y. lipolytica transformantcomprising the vector according to claim
 3. 31. Plasmid pMIVINS.
 32. AY. lipolytica transformant comprising plasmid pMIVINS according to claim31.
 33. A method of producing A14trp proinsulin which comprisesfermenting the Y. lipolytica transformant according to claim 32 in anaqueous nutrient medium comprising assimilable sources of carbon,nitrogen and inorganic salts.
 34. Plasmid pMIVIST.
 35. A Y. lipolyticatransformant comprising plasmid pMIVIST according to claim
 34. 36. Aprocess of producing insulinotropin which comprises fermenting the Y.lipolytica transformant according to claim 35 in an aqueous nutrientmedium comprising assimilable sources of carbon, nitrogen and inorganicsalts.
 37. A method of producing a modified Y. lipolytica LEU2 promoterwhich method comprises:(a) producing a DNA sequence having a 5' end anda 3' end, having homology or substantial homology to a region ofSEQUENCE I.D. NO: 1 and wherein said 5' end is within, but notco-terminus with the 5' end of the promoter region of SEQUENCE I.D. NO:1; (b) producing a vector comprising (i) a DNA sequence wherein the 3'end of the DNA sequence produced according to step (a) is joined to the5' end of a DNA sequence comprising nucleotides of SEQUENCE I.D. NO: 1such that the structural gene for LEU2 is formed and (ii) a sequencecoding for a second Y. lipolytica structural gene; (c) transforming a Y.lipolytica host having a deletion of the LEU2 gene and a mutation ordeletion in the structural gene corresponding to said second structuralgene with the vector produced according to step (b) which vector hasbeen cleaved within the region coding for said second structural gene;(d) selecting Y. lipolytica transformants on a medium containing leucinebut requiring said second structural gene for growth; and (e) screeningthe Y. lipolytica transformants of step (d) for a transformant whichgrows poorly on a medium lacking leucine.
 38. The method according toclaim 37 wherein said second structural gene is URA3.
 39. An isolatednucleotide sequence selected from the group consisting ofGCAGGATCCGAATTCTCTTC CACATAGCAC GGGC (SEQUENCE I.D. NO: 7), GCAGGATCCG AATTCCGTATATATACAAGA GCGTTTGCC (SEQUENCE I.D. NO: 8), GCAGGATCCG AATTCCGTATGTATACAAGA GCGTTTGCC (SEQUENCE I.D. NO; 9), GCAGGATCCG AATTCCGTATAGATACAAGA GCGTTTGCC (SEQUENCE I.D. NO: 10), GCAGGATCCG AATTCCGTGTATATACAAGA GCGTTTGCC (SEQUENCE I.D. NO: 11), GCAGGATCCG AATTCCCACAGATTTTCACT CC (SEQUENCE I.D. NO: 12).40.
 40. Plasmid pNB258.
 41. E. coliATCC
 69353. 42. Plasmid pNB650.
 43. E. coli ATCC
 69354. 44. PlasmidpNB268.
 45. E. coli ATCC
 69355. 46. Y. lipolytica ATCC
 74234. 47.Plasmid pNB308.